The general goal of the project is to provide deeper insight into the structure and function of the heme-copper site in heme-copper oxidases (HCO) and the heme-iron site in nitric oxide reductase (NOR). To achieve our goals, we use a stable, easy-to-produce, and well-characterized heme protein (sperm whale myoglobin, swMb) as the scaffold for making biosynthetic models of HCO and NOR. We have now successfully engineered a CuB site into swMb (called CuBMb), as demonstrated by both spectroscopy and X-ray crystallography. Site-directed mutagenesis and expressed protein ligation (EPL) will be employed to introduce natural and unnatural amino acids, respectively, into the metal-binding sites. Detailed studies of the model proteins will be carried out using spectroscopic tools such as UV-vis, EPR, FTIR, RR, EXAFS, NRVS, and NMR, as well as X-ray crystallography. Kinetic studies will be performed on the protein models to provide insight into the mechanism of the native enzymes. Specific aims are to 1) elucidate the role of CuB in modulating the reduction potentials of the heme-copper center, O2 binding affinity, and reaction mechanism of HCO;2) define the role of heme types (i.e., a/ o or b types) in regulating O2 binding and reduction;3) clarify the role of the covalently linked His-Tyr in modulating the binding affinity and geometry of CuB, the reduction potential of the heme-copper center, and reactivity of HCO;and 4) delineate the role of the metal in the non-heme metal site in determining NOR activity. HCOs catalyze 90% of molecular oxygen reduction in the biosphere. HCO deficiencies or naturally occurring mutations have been linked to Alzheimer's disease, Leigh syndrome, and aging. NOR is one of the key enzymes in the inorganic nitrogen cycle. Well-studied denitrification enzymes may provide potential structural and spectroscopic models for mammalian enzymes that produce and utilize NO in a variety of signal transduction pathways. The work will make important contributions to healthcare, as it will provide a molecular basis for understanding two enzymes important to human health.